The Neural Progenitor Surfaceome
Neural progenitor cells (NPCs) are under clinical investigation for testing their potential for cell-based therapies of neurodegenerative diseases and for the treatment of spinal cord injuries. However, the isolation of pure populations of NPCs suitable for therapeutic applications remains a challenge due to our limited knowledge of specific NPC cell surface molecules useful for flow cytometric or magnetic bead-based isolation and classification[1].
In order to gain a comprehensive view of the NPC surfaceome we applied the Cell Surface Capturing (CSC) technology[2] in combination with label-free quantitative proteomic workflows to create human embryonic stem cells (HUES-7) – derived NPCs, NPCs during neural differentiation and mature postmitotic neurons surfaceome maps. The analysis revealed 680 surfaceome N-glycoproteins which could be relatively quantified between the three selected cell populations. Apart from 53 CD annotated proteins, which are of special interest for further validation due to the availability of antibodies, we identified 627 non-CD annotated proteins. In addition, 1774 NPC N-glycosylation sites were identified which were previously not known or annotated for NPCs.
The comparison of the established surfaceome maps revealed changes in expression of already known cell surface markers like CD133 and CD56 between neuronal precursors and postmitotic neurons, which shows the complementary nature of this relative quantitative approach. Apart from the rediscovery of previously tested molecules we identified a set of unknown and/or untested cell surface markers of neuronal differentiation. Classes of upregulated proteins on NPCs include cell adhesion proteins like cadherins and protocadherins as well as proteins involved in developmental processes like glypicans. Downregulated proteins comprise in comparison to matured neurons Astrotactin-1 and Neurotensin receptor-1. The emergence of a relative quantitative NPC surfaceome barcode provides a basis for their in-depth characterisation and upon separation, for the functional analysis of distinct NPC subsets.
Together, we present surfaceome maps of NPCs, differentiated NPCs and mature postmitotic neurons as a resource for the unbiased selection of cell surface markers for cellular phenotyping, cellular subtype isolation and clinical diagnostics.
Acknowledgement: We acknowledge support from the Czech Ministry of School and Education ME 10044; Institutional Research Concept AV0Z50450515 (IAPG) and European Science Foundation FFG programme #2957.
[1] Gundry RL. et al. Proteomics Clin Appl. 2008 Jun 12;2(6):892-903.
[2] Wollscheid B. et al. Nature Biotechnology, 2009;27:378-386.